Immuno‐ and Correlative Light Microscopy‐Electron Tomography Methods for 3D Protein Localization in Yeast

Compartmentalization of eukaryotic cells is created and maintained through membrane rearrangements that include membrane transport and organelle biogenesis. Three‐dimensional reconstructions with nanoscale resolution in combination with protein localization are essential for an accurate molecular dissection of these processes. The yeast Saccharomyces cerevisiae is a key model system for identifying genes and characterizing pathways essential for the organization of cellular ultrastructures. Electron microscopy studies of yeast, however, have been hampered by the presence of a cell wall that obstructs penetration of resins and cryoprotectants, and by the protein dense cytoplasm, which obscures the membrane details. Here we present an immuno‐electron tomography (IET) method, which allows the determination of protein distribution patterns on reconstructed organelles from yeast. In addition, we extend this IET approach into a correlative light microscopy‐electron tomography procedure where structures positive for a specific protein localized through a fluorescent signal are resolved in 3D. These new investigative tools for yeast will help to advance our understanding of the endomembrane system organization in eukaryotic cells.